Alphaviruses and flaviviruses cause millions of human cases of disease each year, and some of these viruses have been classified as potential biological weapons that might be used by terrorists. Currently, there is a significant need to build a structural foundation to improve our understanding of the alpha- and flavivirus life cycle, as well as to develop antiviral strategies. To answer this call, the Program Project Group proposes to pursue a comprehensive and systematic investigation of the structure and function of the viral proteins within these two groups. The objective of this application is to carry out a comparative structural analysis of the proteins and peptides involved in the viral membrane fusion process. Because the fusion proteins are membrane associated and difficult to obtain, we propose to establish a proteome-scale expression system to identify the best targets for structural analysis and to support parallel studies of several viruses to reveal their common and unique features. In addition to crystallographic studies of individual proteins, we will collaborate with Tim Baker and Michael Rossmann to examine the structural changes of the entire virus that lead to membrane fusion. We will also work with Richard Kuhn and Jim Strauss to design genetic experiments to study the structure-function relationship of the proteins. The proposed multi-disciplinary approach will lead to a greater opportunity to advance our understanding of how alpha- and flaviviruses enter cells and to design fusion inhibitors to prevent viral infection.